Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase.
Methods for the chemical synthesis of 1-butanol are known, such as the Oxo Process, the Reppe Process, and the hydrogenation of crotonaldehyde (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719). These processes use starting materials derived from petrochemicals and are generally expensive and are not environmentally friendly. The production of 1-butanol from plant-derived raw materials would minimize greenhouse gas emissions and would represent an advance in the art.
Methods of producing 1-butanol by fermentation are also known, where the most popular process produces a mixture of acetone, 1-butanol and ethanol and is referred to as the ABE process (Blaschek et al., U.S. Pat. No. 6,358,717). Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is one of the oldest known industrial fermentations, and the pathways and genes responsible for the production of these solvents have been reported (Girbal et al., Trends in Biotechnology 16:11-16 (1998)). Additionally, recombinant microbial production hosts expressing a 1-butanol biosynthetic pathway have been described (Donaldson et al., copending and commonly owned U.S. patent application Ser. No. 11/527,995). However, biological production of 1-butanol is believed to be limited by butanol toxicity to the host microorganism used in the fermentation.
Some microbial strains that are tolerant to 1-butanol are known in the art (see for example, Jain et al. U.S. Pat. No. 5,192,673; Blaschek et al. U.S. Pat. No. 6,358,717; Papoutsakis et al. U.S. Pat. No. 6,960,465; and Bramucci et al., copending and commonly owned U.S. patent application Ser. Nos. 11/743,220, 11/761,497, and 11/949,793). However, biological methods of producing 1-butanol to higher levels are required for cost effective commercial production.
There have been reports describing the effect of temperature on the tolerance of some microbial strains to ethanol. For example, Amartey et al. (Biotechnol. Lett. 13(9):627-632 (1991)) disclose that Bacillus stearothermophillus is less tolerant to ethanol at 70° C. than at 60° C. Herrero et al. (Appl. Environ. Microbiol. 40(3):571-577 (1980)) report that the optimum growth temperature of a wild-type strain of Clostridium thermocellum decreases as the concentration of ethanol challenge increases, whereas the optimum growth temperature of an ethanol-tolerant mutant remains constant. Brown et al. (Biotechnol. Lett. 4(4):269-274 (1982)) disclose that the yeast Saccharomyces uvarum is more resistant to growth inhibition by ethanol at temperatures 5° C. and 10° C. below its growth optimum of 35° C. However, fermentation became more resistant to ethanol inhibition with increasing temperature. Additionally, Van Uden (CRC Crit. Rev. Biotechnol. 1 (3):263-273 (1984)) report that ethanol and other alkanols depress the maximum and the optimum growth temperature for growth of Saccharomyces cerevisiae while thermal death is enhanced. Moreover, Lewis et al. (U.S. patent Application Publication No. 2004/0234649) describe methods for producing high levels of ethanol during fermentation of plant material comprising decreasing the temperature during saccharifying, fermenting, or simultaneously saccharifying and fermenting.
Much less is known about the effect of temperature on the tolerance of microbial strains to 1-butanol. Harada (Hakko Kyokaishi 20:155-156 (1962)) discloses that the yield of 1-butanol in the ABE process is increased from 18.4%-18.7% to 19.1%-21.2% by lowering the temperature from 30° C. to 28° C. when the growth of the bacteria reaches a maximum. Jones et al. (Microbiol. Rev. 50(4):484-524 (1986)) review the role of temperature in ABE fermentation. They report that the solvent yields of three different solvent producing strains remains fairly constant at 31% at 30° C. and 33° C., but decreases to 23 to 25% at 37° C. Similar results were reported for Clostridium acetobutylicum for which solvent yields decreased from 29% at 25° C. to 24% at 40° C. In the latter case, the decrease in solvent yield was attributed to a decrease in acetone production while the yield of 1-butanol was unaffected. However, Carnarius (U.S. Pat. No. 2,198,104) reports that an increase in the butanol ratio is obtained in the ABE process by decreasing the temperature of the fermentation from 30° C. to 24° C. after 16 hours. However, the effect of temperature on the production of 1-butanol by recombinant microbial hosts is not known in the art.
There is a need, therefore, for a cost-effective process for the production of 1-butanol by fermentation that provides higher yields than processes known in the art. The present invention addresses this need through the discovery of a method for producing 1-butanol by fermentation using a recombinant microbial host, which employs a decrease in temperature during fermentation, resulting in more robust tolerance of the production host to the 1-butanol product.